Plant Pathology 602Plant-Microbe Interactions

Sophien Kamounkamoun.1@osu.eduThe Ohio State UniversityOhio Agricultural Research & Development Center

Lecture 2Molecular methods for studying host-pathogen interactions I

n Refresher on gene expression

n Strategies for molecular study of plantpathogens and their hosts

Plant Pathology 602Plant-Microbe Interactions

Outline - lecture 2

n Refresher on gene expression

n Strategies for molecular study of plantpathogens and their hosts

Plant Pathology 602Plant-Microbe Interactions

Outline - lecture 2

Refresher on gene expression - DNA: The stuff of life

Refresher on gene expression

Gene

Refresher on gene expression

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Refresher on gene expression

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Refresher on gene expression

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Refresher on gene expression

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Refresher on gene expression Central dogma of molecular biology

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Replication

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DNA cloningAmplification of DNADNA sequence

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DNA cloningAmplification of DNADNA sequence

cDNAsynthesized in vitro

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DNA cloningAmplification of DNADNA sequence

DNAcloning

cDNAsynthesized in vitro

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DNA cloningAmplification of DNADNA sequence

DNAcloning

Protein PurificationBiochemical analyses

cDNAsynthesized in vitro

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DNA cloningAmplification of DNADNA sequence

DNAcloning

Protein PurificationBiochemical analyses

cDNAsynthesized in vitro

Refresher on gene expression

n Structurally simpler

n Cells do not contain nucleus

n Genomic DNA generally

naked and circular

n Genes are generally present

in single sets (haploidy)

n Structurally more complex

n Cells contain a true nucleus

n DNA contained in

chromosomes in nuclei

n Cell organelles

(mitochondria, chloroplasts

etc…)

n Genes are present in two or

more sets (diploidy)

prokaryotes eukaryotes

Refresher on gene expressionTechnical challenges in the molecular geneticstudy of eukaryotes

n Structurally more complex

n DNA transformation can be limiting (cell walletc…)

n Large genomes (lots of noncoding sequences,gene families...)

n Diploid organisms (more than one gene to disrupt)

n Refresher on gene expression

n Strategies for molecular study of plantpathogens and their hosts

Plant Pathology 602Plant-Microbe Interactions

Outline - lecture 2

Strategies for molecular study of plantpathogens and their hostsGenetic approach

n Many methods are centered around a(molecular) genetic approach

n Basis of the genetic approach is to start withtwo individuals/populations that differ inphenotype and identify the genes thatdetermine the difference

n Variation could be natural or artificiallygenerated by mutagenesis

Genetic approach- Natural variation

Pathogen

Plant

ResistanceDisease

Strain A Strain B

Genetic approach- Mutagenesis

Pathogen

Plant

ResistanceDisease

Wild-type Mutant

X

Strategies for molecular study of plantpathogens and their hostsBiochemical approach

n A protein/secondary metabolite with aparticular property can be purified from thepathogen/plant

n The amino-acid sequence of the protein canthen be determined and the correspondinggene isolated

n Example: toxin or plant cell wall degradingenzymes isolated from pathogens

Biochemical approach

Pathogen

Plant

Toxic effectDisease

Strain A

Strategies to isolate pathogen genesinvolved in disease (virulence/avirulence)

n Gene disruption

n Gene silencing

n Map-based cloning

n Differential gene expression

n Biochemical approach

n Genomics (random sequencing of DNA)

Strategies to isolate plant genes involved indisease (resistance/susceptibility)

n Mutagenesis (transposon and others)

n Map-based cloning

n Differential gene expression

n Biochemical approach

n Candidate genes (similarity based)

n Model plants (Arabidopsis, rice)

Gene transfer = DNA transformation Fungi/oomycetes

n Proroplasts or zoospores

n Liposome mediated

n Electroporation

n Particle bombardment

n Agrobacterium-mediated

Reporter genes (ex: GFP or GUS)

n GFP: green fluorescent protein

n GUS: beta glucuronidase

Reporter genes (ex: GFP or GUS)

n GFP: green fluorescent protein

n GUS: beta glucuronidase

n Constitutive promoters

n Inducible promoters

promoter-reporter gene

mRNA

Gene transfer = DNA transformation Plants

n Stable or transient

n Agrobacterium-mediated

n Particle bombardment

n Transgene - exogenous recombinant DNAfragment(s) introduced into plants

n Transgenic plants - plants that containexogenous recombinant DNA, also commonlycalled Genetically Modified Organisms or GMOs

Gene transfer into plant cells

How are transgenic plants generated?

BACTERIA PLANT CELLWITH TRANSGENE

TRANGENIC PLANT

n Agrobacterium - The

natural genetic engineer!!!!

n A bacterium that can

naturally transfer genes into

plants

Gene transfer into plant cells

n Particle bombardment- A method for introducingDNA into plant cells usinga gene gun

Gene transfer into plant cells

http://www2.oardc.ohio-state.edu/plantranslab/

n Totipotency - unique ability of isolated plantcells to regenerate into a whole plant

Plant regeneration

http://www2.oardc.ohio-state.edu/plantranslab/

Transient transformation assays in plants

n Agroinfiltration

n Virus based (example Potato Virus X)

n Transformation of protoplasts

(electroporation)

Agroinfiltration AssayExample: HR elicitors

T-DNA

Vir

Chromosome

35S promoter-elicitor

LB RB

A. tumefaciens

Expression of HR elicitor genes fromthe Potato Virus X genome

166K 25K 8K CP12K

PVX

166K 25K 8K CP12K

PVX-INFelicitor

Gene disruption or gene knockout

n Gene disruption can be obtained by insertinga piece of DNA inside the target genecreating a “disruption” or mutation

n Inserted DNA is generally an artificiallyengineered fragment and usually contains agene for antibiotic resistance for selection

n Insertion can be random or can be targetedthrough the process of homologousrecombination

Gene disruption or gene knockout

Antibiotic resistance

Target gene Disrupted geneMUTANT!!!

Gene disruption or gene knockoutChallenges for eukaryotes

n Most fungal species are haploid but in diploidspecies disruption of the two copies isnecessary to obtain a null mutant

n DNA transformation can be limiting

n Rates of homologous recombination varybetween organisms (example: very low frequency

in Phytophthora)

Gene silencing or RNAi (RNA intereference)

n Introduction of a gene in an antisense (wrong)orientation or sometimes even in a senseorientation triggers a gene silencing response inthe eukaryotic cell

n “Foreign” DNA is silenced that is the gene is notexpressed anymore or the RNA gets degraded

n If the “foreign” DNA is similar to a gene of thetransformed organism, silencing will affect boththe transgene and the endogenous genes thusresulting in a mutant phenotype

Gene silencing - Example: Virus inducedGene Silencing (VIGS)phytoene desaturase in tobacco results in bleaching

Source: D. Baulcombe lab

TRV TRV::sgt1

Virus Induced Gene Silencing tocharacterize plant response to elicitors Map-based cloning

n If individuals with different phenotypes are availablethen genetic crosses can be set up and segregation ofthe differing genes can be noted

n Molecular markers (mutation scattered throughout thegenome and identified using molecular tools such asrestriction enzyme digests or PCR) can be identifiedin this segregating population

n Markers that are genetically linked to the segregatinggene can be used to identify the target gene

Map-based cloning - genetic map

1 2 3 4

Differential gene expression

n Pathogen and plant genes that are highlyexpressed during infection are likely to beinvolved in the infection process

n Patterns of gene expression are revealed by thepopulations of mRNA present in the cell

n Methods to compare and subtract different RNA(cDNA) populations exist and can be applied tothe identification of infection induced genes

The interaction transcriptome

Genomics (random sequencing of DNA)

n Genomics or random sequencing of genomicDNA and/or cDNA can be applied to the study ofplant-microbe interactions

n Various computer analyses of the sequencedgenes can be used to identify candidate genes(bioinformatics)

n Functional genetic assays need to be performedto test the function of the candidate genes(functional genomics)

Example: cDNA sequencing (ExpressedSequence Tags)

ORFATG TAG5’UTR 3’UTR

AAA

The functional genomics paradigm

Sequence Candidategenes

Phenotype

bioinformatics Functional assay

Paradigm shifts in biological researchAdapted from Peltonen and McKusick, Science 291:1224

Structural genomics --> Functional genomicsGenomics --> ProteomicsMap-based --> Sequence-based gene discoveryMonogenic traits --> Multifactorial traitsAnalysis of one gene --> Analysis of multiple genes

in gene families, pathways, or systems

Gene action --> Gene regulationEtiology (mutation) --> Pathogenesis (mechanism)One species --> Several species

n Short generation time

n Small genomes

n Genome sequences available

n Powerful mutagenesis tools

n DNA transformation and functionalassays available

n Resistant to diverse pathogens

Model plants (Arabidopsis, rice) Glossary of genetic terms

n http://www.weihenstephan.de/~schlind/genglos.html

n http://www.genome.gov/glossary.cfm

The interaction transcriptome